Process for extraction and standardization of pharmaceutical quality tinctures and extracts from herbal products

ABSTRACT

The present invention relates generally to herbal materials and methods for making such materials in medicinally useful and pharmaceutically acceptable forms. Particularly, the present invention relates generally to  Hydrstatis canadensis  (goldenseal) materials and methods for making such materials in medicinally useful and pharmaceutically acceptable forms. More particularly, the present invention relates a process which allows the precise standardization of at least two marker compounds, berberine and hydrastine, in both hydro-alcoholic tinctures and solid extracts in the processing of goldenseal materials to produce extracts which qualify as pharmaceutical grade compositions which are suitable for use in clinical or veterinary settings to treat and/or ameliorate diseases, disorders or conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/549,747, filed Mar. 3, 2004. The entire disclosure of this prior application is hereby incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support under Grant No. 2003-33610-13119, awarded by United States Department of Agriculture. The United States Government has certain rights in this invention.

SEQUENCE LISTING OR PROGRAM

Not Applicable

BACKGROUND OF THE INVENTION

1. Field of the Invention

The lack of standard levels of pharmacologically active compounds in natural botanical products has resulted in a reluctance by health care providers to prescribe these products for their patients. This problem is compounded by the fact that even when botanical products are standardized to specific levels of a marker compound, variations in the content of other oftentimes unknown compounds caused by a wide range of factors such as soil temperature and pH, air temperature, rainfall, and genetics, results in unpredictable biological activity. Additionally, raw material integrity is vital to the production of a safe and effective product. Therefore, this invention is directed to a method of assuring reproducibility of an extraction process. The present invention is also directed to a method of reproducibly extracting a pharmacologically active mixture of chemical components from a biological source, particularly a plant source. Furthermore, the present invention is directed to a method of manipulating the pharmacologically active compound concentration levels within said pharmacologically active mixture of chemical compounds relative to certain other unknown compounds within the mixture. Thus, the method delivers a pharmaceutical grade product with a desired level of bioactivity without adulteration of any kind. The present invention also reveals a composition of botanical material with enhanced biological activity when compared with the individual parts or the sum of the parts.

2. Background of the Invention

Plants have been, and continue to be, the primary source of a wide variety of medicinal compounds. For centuries, various forms of botanically derived materials have been used to treat countless different ailments. The botanical materials have typically been in the form of powders made from one or more plants or plant parts or extracts derived from whole plants or selected plant parts. These powders and extracts are, for the most part, complex mixtures of both biologically active and biologically inactive compounds.

In modern day, herbal medicine, although gaining some acceptance in Western society, still faces several specific challenges. First, in the opinion of many highly trained medical practitioners, there is the view that herbal medicine lacks sufficient scientific support data in our highly technical and science oriented society. Secondly, there is concern about which components of an herbal remedy are pharmaceutically effective. Furthermore, the question arises as to the concentrations or dosages present of such pharmaceutically effective components of herbal remedies. In short, traditional medical practitioners are concerned with a lack of both qualitative and quantitative standards for herbal medications. Such a lack of standardization is viewed as hindering the ability to prescribe and adjust dosages of such nontraditional or herbal medications. The lack of such standardization has also lead to a reluctance on the part of regulatory agencies in funding further investigation and acceptance of such nontraditional medications.

Although not meeting some of the criteria of Western traditional medicine, such herbal compositions are known to be quite effective in treatment of a variety of maladies with little or no side effects. In part, the pharmaceutical activity in many instances is attributable not only to the presence of specific biologically active compounds but also to a synergistic effect resulting from the combination of two or more chemical components present in the herbal mixture.

Since herbal treatments, defined as both herbal medications and biologically enhancing herbal compositions, are derived from plants, the chemical composition of such herbal treatments varies according to a number of factors, not the least of which are the genetic composition and growing conditions in which the plant is produced as well as the harvest conditions and isolation of the active components of the plant.

Accordingly, biological variants of a particular plant may typically be expected to produce significant variations in quantities of particular chemical components found in the plant. Likewise, even in the same biological variant of a plant, differences in soil, moisture and other growing conditions may significantly affect the quantities of specific chemical components produced by the plant.

The vast majority of the raw material used to produce botanical products is currently collected form the wild. Wild collected plants are, by definition, produced outside of a controlled environment. This has presented a unique problem for manufacturers of botanical products desiring the control, reproducibility, and standardization that are required of pharmaceuticals. This problem is due primarily to the plurality of components contained in an herbal medicine and the large variation in composition and potency due to the growing, harvesting and processing conditions. Therefore it is desirable to standardize these growing conditions to the extent possible.

Finally, the manner in which a plant is processed can drastically influence the relative proportions and total amounts of specific chemical components isolated from the plant. Thus, such steps as harvesting, storage, reduction in particle size, expression of liquid components and extraction all determine the proportions and amounts of chemical components and hence the pharmaceutical activity of the isolated product.

Considering the many factors which influence the composition and pharmaceutical activity of herbal compositions, it is desirable to employ methods which result in the standardization of herbal compositions both with respect to the chemical compositions thereof and the pharmaceutical activity of such chemical mixtures. In addition, it is desirable to standardize the processing conditions in order to obtain such standardized herbal compositions. Furthermore, being able to accurately determine and compare the compositions of biological mixtures, particularly plant or herbal mixtures, would allow processing conditions to be controlled to obtain reliable pharmacological activity. With such methods available to the scientific community, not only would physicians be able to prescribe specified dosages of herbal compositions with confidence, but herbal composition “manufacturers” would achieve predictable pharmaceutical activity of such mixtures, improved quality control and the ability to differentiate herbal mixtures from varying sources.

Pharmaceutical grade botanical products are advantageous in that they allow careful tracking of the effects of individual compounds, in treatment protocols. Further, the dosage of the drug can be carefully controlled to provide relatively predictable medicinal action. The potential benefit provided by the use of herbal product as medicine is believed by many industry experts to be outweighed by the clinical risks associated with the absence of standard levels of biologically active materials from natural plants. In that respect, herbal products are not well characterized or controlled in a clinical setting.

The present invention is a significant advancement in the field of making herbal extracts for medicinal purposes, providing precise levels of both berberine and hydrastine while at the same time maintaining USDA National Organic Program (NOP) organic certification. The invention offers tremendous benefits since the extract is standardized to biological activity rather than just a chemical marker. This allows for the use of exact quantities of the extract necessary to produce the desired pharmacological activity instead of potentially toxic high doses or ineffective low doses produced by current methods. The invention also permits the manipulation and/or removal of certain pharmacologically active compounds from the extract thereby allowing more precise calculation of the contribution of each compound's activity to that of the whole and permits various ratios of the compounds in the final product based on the results of certain bioassays. In addition, pre-standardization by cultivation and harvesting practices results in higher concentrations of pharmacologically active compounds in the raw material which translates into reduced processing to produce a given amount of extract. Maintenance of USDA NOP certification throughout the production process provides third party certification that the product has been produced in a sustainable manner, important due to many medicinal plant's endangered status, and that no potentially toxic materials such as herbicides, pesticides, or artificial fertilizers have been used.

In one object of this invention, a method was developed for standardizing the levels of biologically active materials from natural plants including, but not limited to, herbal plants. In a more preferable object of this invention, the method is suitable for producing pharmaceutical grade natural products standardized to a specific level of bio-activity from Hydrastis canadensis Linn. (goldenseal) and other berberine and/or hydrastine containing plants. In a preferable object of this invention, the method disclosed is suitable for the precise standardization of marker compounds generally recognized as indicators for the quality of goldenseal. In a more preferable object of this invention, the marker compounds are berberine and hydrastine.

Background Information Regarding Goldenseal

Goldenseal belongs to the Family Ranunculaceae and is a small hairy perennial which in a natural environment generally emerges in early spring (mid-March to early May) and dies down in mid-August to late-September. The root system is composed of a bright yellow horizontal rhizome, 2 to ¾ inch thick, marked by cup-like depressions where the annual stem falls away. Mature plants (at least 3 years old) are 6-14 inches tall and have two or more hairy stems usually ending in a fork with two leaves. The 5-7 lobed, palmate, double-toothed leaves are 3-12 inches wide and 3-8 inches long. After emergence in early spring, flower buds quickly develop and small inconspicuous white flowers open as the leaves unfold. Plants started from seed usually flower the third or fourth year. Each plant can produce a single, green raspberry like fruit which turns red and ripens in July (Davis, 2000).

Goldenseal is one of the most popular medicinal herbs in the US and has been wild collected from the forests of Eastern North America for hundreds of years. Its historical range extended from Vermont to Georgia to Arkansas to Minnesota (Foster, 1990). Goldenseal's popularity has resulted in it being overcollected from the wild. Goldenseal is listed as an Endangered Species in Georgia, North Carolina, Vermont, Connecticut, Massachusetts, and Minnesota. The USFWS has stated that goldenseal is either endangered, threatened, imperiled, rare, or uncommon in all 27 U.S. states which have native populations (USFWS, 1997). The US government, in 1997, backed a proposal to place Goldenseal on the Convention on International Trade in Endangered Species (CITES) Appendix List II, which was approved. The CITES listing requires the goldenseal produced for export be cultivated for minimum of four years. The present invention addresses this problem through the development of a “wild simulated” production system for goldenseal.

The alkaloids berberine and hydrastine are commonly used by industry as markers to indicate the quality of raw goldenseal although the most common level of consumer product assurance is that of a specified percentage of total alkaloid content, unspecified as which alkaloid or how much of each. These two compounds are also thought to be the primary bio-active compounds in the plant. (Abourashed, 2001, Govindan, 1999) Proposed United States Pharmacopoeia (USP) standards for dried goldenseal root and rhizome are not less than 2.0% hydrastine and not less than 2.5% berberine.

The alkaloid content variability of both raw goldenseal and consumer products currently on the market was clearly demonstrated in Govindan (1999), where ten samples (eight—raw powdered material, GS-1 through GS-8 and two—powdered material from off-the-shelf capsules, GS-9 and GS-10) were analyzed using Thin-Layer Chromatography (TLC). Five of the samples contained both berberine and hydrastine, four contained only berberine, and one contained neither. These results were verified by High Pressure Liquid Chromatography (HPLC) (Govindan, 1999). Hydrastine is unique to goldenseal among North American plants therefore the absence of hydrastine is a strong indication that the material purported to be goldenseal root and rhizome is probably not goldenseal at all. Extremely low quantities of hydrastine are also indicative of adulteration with other berberine containing plants such as various Berberis species. Discovery of palmatine in purported goldenseal material, common in other berberine producing plants but absent in goldenseal, is a definitive indication of adulteration (Weber, 2003). In addition only three of the ten samples tested approximated the profile of the raw goldenseal used by the National Toxicological Program for their studies (MRI, 2001). Our 2003 harvest (SHF-1) had the second highest combined hydrastine/berberine total of all the samples listed. See table 1 below.

The present invention provides both a method of standardized processing procedures and of obtaining biological compositions having a desired level of pharmaceutical activity from plants containing berberine and/or hydrastine, preferrably goldenseal. The present invention also permits the isolation of biological compositions or components, and in particular herbal compositions or components, having high, or the highest pharmacological activity obtainable by a specific process, such as extraction.

TABLE 1 Results of TLC and HPLC analysis of 10 goldenseal samples (GS-1-GS-10)^(a), National Toxicological Program Sample(NTP-1)^(b), one commercial product analyzed in Weber, et al, (1999)^(c) and Sleepy Hollow Farms 2003 harvest. Hydrastine Berberine Hydrastinine Sample TLC HPLC(%) TLC HPLC(%) TLC HPLC(%) GS-1 +++ n.a. +++ n.a + n.a. GS-2 +++ 2.68 +++ 4.27 + n.a. GS-3 − − ++ 0.56 + n.a. GS-4 +++ 2.34 +++ 3.48 + n.a. GS-5 +++ 3.22 +++ 4.54 + n.a. GS-6 + 0.60 ++ 0.56 − n.a. GS-7 − − ++ 0.28 − n.a. GS-8 − − − − − n.a. GS-9 − − +++ 1.51 − n.a GS-10 − − +++ 5.31 − n.a. NTP-1 n.a. 3.02 n.a. 3.45 n.a. n.a. Weber-1 n.a. 1.30 n.a. 1.90 n.a. n.a. SHF-1 n.a. 3.31 n.a. 3.96 n.a. n.a. ^(a)Relative amounts based on the visual estimation of the size and intensity of spots on TLC: dark spot(+++); medium intensity spot(++); low intensity spot(+); not visible(−); not analyzed(n.a.)(Govindan, 1999). ^(b)(MRI, 2001) ^(c)(Weber, et al., 1999) Use of Goldenseal and/or its Isolated Alkaloids Berberine/Hydrastine

Goldenseal was first used by Native Americans to treat wounds, ulcers, digestive disorders, and skin and eye ailments. Over the years goldenseal has been used to treat a variety of digestive and hemorrhagic disorders. It is thought to possess antiseptic, astringent, and hemostatic qualities when applied topically. It is thought by some that goldenseal is effective in the treatment of diarrhea, hemorrhoids, disorders of the genito-urinary tract, upper respiratory inflammation and congestion, mucous membrane inflammation, eczema, pruritus, otorrhea, tinnitus and congestion/inflammation of the ear, and conjunctivitis, as well as for cancers, particularly of the ovary, uterus, and stomach. Goldenseal has been used as a tonic, antiperiodic, diuretic, and as a vaginal douche. It is commonly consumed in capsules, liquid herbal extracts, and as an herbal tea.

Although at least 10 isoquinoline alkaloids have been identified in goldenseal, berberine is considered to be the primary pharmacologically active compound in goldenseal. Berberine's most common historic and clinical uses include bacterial diarrhea, intestinal parasites, and ocular trachoma infections. Berberine has been shown to exhibit significant antimicrobial activity against a variety of bacteria, fungi, protozoans, helminths, chlamydia, and viruses (Birdsall, 1997). Some berberine containing plants have been shown to produce substances which, by themselves, are completely without antimicrobial activity however, when used in conjunction with berberine, they enhanced the activity of berberine by as much as 2,500 times (Stermitz, 2000, Tegos, 2002). In addition to this antimicrobial activity, berberine has been found to have numerous pharmacological effects including antagonism of the effect of cholera and E. coli heat stable enterotoxin (Sack, 1982), delay of small intestine transit time (Eaker, 1989), inhibition of intestinal secretions (Zhu, 1983), significantly inhibit spontaneous peristalsis in the intestine (Birdsall, 1997), inhibition of smooth muscle contraction (Tai, 1981), potent inflammation inhibitory activity (Yesilada, 2002), inhibition of cyclooxygenase-2 (COX-2) transcriptional activity in a dose and time dependent manner (Fukuda, 1999), and inhibition of II-8 production in rectal mucosa in rats (Zhou, 2000). Furthermore, berberine has been shown to produce significant cardiovascular, cholesterol reduction, MAO inhibition, and antidiabetic activities. Berberine is considered to be a non-antibiotic anti-diarrheal drug (Baird, 1997).

Several recent studies have been conducted regarding the in vitro effectiveness of the antimicrobial constituents of goldenseal crude extract and its isolated alkaloids against a variety of oral and digestive tract pathogens including Streptococcus mutans, Fusobacterium nucleatum, and 15 strains of Helicobacter pylori. Results indicated the crude extract and isolated berberine to be very active against these pathogens (Hwang, 2003, Mahady, 2003).

In a 2001 study, the antibacterial activity of crude goldenseal extract and the isolated alkaloids berberine, hydrastine, and canadine were evaluated against five strains of microorganism: Staphylococcus aureus (ATCC 25 993 and ATCC 6538P), Streptococcus sanguis (ATCC 10 556), Escherichia coli (ATCC 25 922), and Pseudomonas aeruginosa (ATCC 27 853). The results of this study were reported to provide a rational basis for the traditional antibacterial use of goldenseal (Scazzocchio, 2001).

Berberine, isolated from the roots of goldenseal, was demonstrated to be responsible for the significant activity of goldenseal extract against multiple drug resistant Mycobacterium tuberculosis. (Gentry, 1998)

A 1998 study conducted at Iowa State University entitled Botanicals for Pigs—Goldenseal compared goldenseal's use as a natural antimicrobial agent in nursery pigs with Mecadox. The study reported that growth of the pigs on a 1% goldenseal diet were often not statistically different from the Mecadox controls.

One significant thread common to the above studies is the fact that in each instance the activity of the crude extract was shown to be equal to or greater than the activity of isolated berberine. In one particular instance, Hwang (2003), the crude extract of goldenseal root and rhizome was demonstrated to have comparable activity to the isolated berberine even though the crude extract only contained 0.02% berberine, 1/5000th the concentration of the isolated berberine. All other fractions and combinations of alkaloids produced only minimal activity and can not possibly account for the increased activity of the crude extract. This indicates there are other unknown compounds in goldenseal root and rhizome which are inactive by themselves but significantly enhance berberine's activity when combined. Accordingly standardization to specific quantities of berberine in a goldenseal product will most likely produce varying pharmacological activity due to year to year variations in environmental conditions which produce not only fluctuating amounts of berberine but also the unknown compounds.

This invention reveals a method of manipulating the concentration levels of goldenseal's primary marker compounds within the final product without adulteration of any kind. In addition, the invention goes a step further by revealing a method of quantifying the effects of the unknown compounds in goldenseal then using the aforementioned method to produce a goldenseal product which produces a predictable level of pharmacological activity.

SUMMARY OF THE INVENTION

Accordingly, it is a first object of the present invention to provide an improved method for isolating or obtaining herbal extracts.

Another object of the present invention is a method of manufacturing herbal extracts.

A still further object of the present invention is a method for isolating or obtaining herbal extracts that are economically attractive. In one aspect of the invention, the herbal extract is used to treat human and non-humans. Non-human, include but not limited to, farm animals such as chickens, pigs, cows, fish and others.

Yet another object of the invention is a method that produces herbal extracts with more predictable levels of pharmacological activity over prior art techniques.

-   -   One other object of the present invention is a method of making         high concentration extracts in lower unit doses for patient use.         Other objects and advantages will become apparent as a         description thereof proceeds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Diagram of production Process.

FIG. 2. Relative Recoveries from three Methods of Drying One Pound of Fresh Goldenseal.

FIG. 3. Hydrastine/Berberine Content Goldenseal after Three Methods of Drying.

FIG. 4. Net effect of drying and grinding goldenseal on gross alkaloids extraction compared with fresh material.

FIG. 5. Alkaloid Extraction as a function of Alcohol Concentration.

FIG. 6. Combined hydrastine/berberine recovered from five different solvent levels over three extractions.

FIG. 7. Goldenseal Extraction Cost Optimization.

DETAILED DESCRIPTION OF THE INVENTION

Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

In this specification and the appended claims, the singular forms “a,” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

The term “composition”, as used herein, refers to a mixture of components. As used herein, “components” refers to chemical compounds, salts of such compounds, complexes and other molecular and ionic species found in nature.

The term “herbal”, as used herein, refers to plant substances materials and/or parts.

The term “pharmaceutical grade” when used in this specification means that certain specified biologically active and/or inactive components in a botanical drug must be within certain specified absolute and/or relative concentration range and/or that the components must exhibit certain activity levels as measured by a disease-, disorder- or condition-specific bioactivity assay. The disease, disorder or condition may afflict a human or an animal.

As will be understood by those skilled in the art, the term “pharmaceutical grade” is not meant to imply that the botanical drug is applicable only to products which are regulated for example, those provided under prescription, i.e., “Rx” products or over the counter, i.e., “OTC”. The term is equally applicable to products provided Rx, OTC or as a dietary supplement, i.e., “DSHEA”.

All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the subject components of the invention that are described in the publications, which components might be used in connection with the presently described invention.

The information provided below is not admitted to be prior art to the present invention, but is provided solely to assist the understanding of the reader.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

One criticism traditionally charged to the use of herbal products as medicines is the absence of standard levels of biologically active materials from natural plants. The present invention provides both a method of obtaining standardized biological compositions having a desired level of pharmaceutical activity and a method of standardized processing procedures. The present invention also permits the isolation of biological compositions or components, and in particular herbal compositions or components, having high, or the highest pharmacological activity obtainable by a specific process, such as extraction.

In one aspect of this invention, the methods of the present invention used for standardization of a biologically or pharmacologically active mixture of chemical components obtained from a biological source, preferably a plant, involve initially conducting a plurality of different processes using a plurality of samples from the same biological source, preferably plant source, most preferably goldenseal, to produce a plurality of crude extracts. Physical and/or chemical tests are then performed on the crude extracts to provide qualitative and, in most instances, quantitative information regarding the chemical component(s) of the crude extracts. In one aspect of the invention, the crude extracts from these processes are combined, sampled, analyzed and then concentrated to a predetermined point. In one aspect of the invention, the samples are analyzed using an HPLC or alike. In one aspect of the invention, the samples are concentrated using an evaporator system in conjunction with vacuum or a like.

In one aspect of this invention, a method is provided for processing dried or fresh goldenseal produced from either wild collected, conventional cultivated, or certified organic sources or a combination thereof in order to produce a hydro-alcoholic tincture and/or a solid extract with consistent and predictable amounts of at least two goldenseal quality marker compounds, berberine and hydrastine as well as the bioactivity thereof. In one aspect of this invention, the preferred source is certified organic plant source which yields a 100% certified organic product as defined by the USDA NOP.

When the source of the mixture of the chemical components is a plant source, such as a mixture used in an herbal medication or composition, typical processes may include methods of harvesting, methods of storage, methods of expressing liquid components and, preferably, methods of extraction of chemical components, most preferably the chemical components responsible for pharmacological activity. A method is chosen for a particular process and variables are changed, when possible, one at a time to produce a plurality of method products.

The present invention is expected to have most widespread application to plant sources, in particular goldenseal.

In one aspect of this invention, the inventor has developed a process which allows the precise standardization of at least two marker compounds generally recognized as indicators for the quality of Hydrastis canadensis (goldenseal) which are believed to be the primary biologically active constituents, berberine and hydrastine, in both hydro-alcoholic tinctures and solid extracts of goldenseal. The process is suitable for use in, but not limited to, conventional and 100% certified organic production systems. This invention also reveals compositions of berberine and hydrastine, within the context of a whole plant goldenseal tincture or extract, which produce enhanced immune system activity in humans and/or animals. Furthermore, this invention reveals compositions of berberine and hydrastine, within the context of a whole plant goldenseal tincture or extract, which suppress growth and virulence factors of certain human, animal, and plant pathogens including but not limited to, Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Candida albicans, Candida glabrata, Candida tropicalis, Candida utilis, Chlamydia trachomatis, Corynebacterium diphthenae, Cryplococcus neoformans, Entamoeba histolytica, Escherichia coli, Fusobacerium nucleatum, Giardia lamblia, Helicobacter pylori, Klebsiella pneumoniae, Leishmaniasis sp., Microsporum gypseum, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Shigella boydii, Sporoinchum schenkii, Staphylococcus aureus, Staphylococcus albus, Streptococcus sanguis, Streptococcus mutans, Strepococcus pyogenes, Trichomonas vaginalis, Trichophyton metangrophytes, and Vibrio cholera.

In the method of the invention, the extraction solvent is preferably an ethanol and water mix but may be other solvents. A protocol using a known starting weight of the herbal material, a known volume of finished product to be obtained and a percentage of alcohol in the finished extract based on the herb to be processed can be used to initiate and guide the method.

Extracts, tinctures or the like produced by the inventive method can be used as is or combined with other extracts for medicinal purposes. In addition, one or more extracts can be utilized to make tonics for medicinal use.

The separatory procedure employed is preferably a chromatographic procedure such as high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), high pressure thin layer chromatography (HPTLC). Capillary electrophoresis (CE) and UV/Vis spectrophotometric methods can also be used.

Preferable detection systems in the case of HPLC, include absorbance and fluorometric spectroscopy, refractive index, electrochemical methods, evaporative light scattering, electrospray mass spectrometry, or a combination of these.

The botanical materials for the process can be fresh, fresh dried, or dried. The part of the plant used as the raw material can vary as well. Certain plants have most of the active principles in the roots while others may have the active principles in the leaves, and some have active principles in both. The present method is intended for use with all parts of the targeted plant, including flowers, leaves, stems, and roots combined in calculated percentages to produce pre-standardized raw material for extraction purposes.

The botanical material may be processed to form an aqueous or organic extract of the whole plant or a selected part of the plant. The botanical material can also be processed in whole or part to form a powder. Many of the botanicals of interest are commercially available as powders, aqueous extracts, organic extracts or oils. In one embodiment, extracts of the plant material are preferred because they are easier to dissolve in liquid pharmaceutical carriers. However, powdered plant materials are well-suited for many applications where the drug is administered in solid form, e.g., tablets or capsules. Such methods of producing these products are well known to those of skill in the art. Furthermore, many of the plant materials and/or extracts are available commercially however, plant material cultivated under USDA NOP certified wild simulated conditions is the preferred source. As examples of the cultivation, processing and extracting of botanicals the following examples are provided.

For a typical root, it may be sliced, frozen or pulverized. If powdered it is then shaken with an appropriate solvent and filtered. Alternatively, the following methods are used: the root is homogenized, ethanol extracted and filtered. The initial solvent extract from the methods above may be further extracted using liquid/liquid extraction with an appropriate solvent. Botanicals may also be processed as a paste or powder which may be cooked.

A variety of solvents may be used in the extraction process; for example acetone, acetonitrile, dichloromethane, ethyl acetate, ethanol, hexane, isopropanol, methanol, other alcohols, water, and supercritical carbon dioxide and mixtures thereof.

The dried material may be prepared in a variety of ways including freeze-drying, drying via microwave, cooling with liquid nitrogen and pulverizing; or air-drying in the shade, or with forced heated air at about 40° C.

The following is an example of a process to produce a 100% certified organic goldenseal liquid extract. The example is intended only for illustration purposes of the several aspects of the invention.

Raw goldenseal is available from a variety of sources. Wild collected, which accounts for approximately 95% of the raw goldenseal currently on the market, is the least desirable source due to goldenseal's endangered status and unknown contamination and/or adulteration with undesirable substances. Cultivated goldenseal is the preferred source with wild simulated cultivation in a natural forest environment in conformance with the USDA NOP (certified organic) most preferred. USDA NOP standards are incorporated in this specification in their entirety by reference.

Since goldenseal is primarily wild collected only limited information is available regarding optimal cultivation and almost none regarding optimal post-harvest handling methods. Wild simulated certified organic cultivation in a natural forest environment is the most preferred source of raw material therefore it was necessary to survey locations where goldenseal is native to determine optimal soil chemical characteristics for plantation establishment. Table 2 lists the soil chemical profiles for three native goldenseal sites as well as beginning soil chemical characteristics for a proposed certified organic goldenseal cultivation site before and 1 year after soil amendments were added.

Native site # 1 was located on public land in Walker county, Ga. on the eastern escarpment of the southern portion of the Cumberland Plateau. We were accompanied to the site by the Program Botanist of the Georgia Department of Natural Resources Plant Protection Program. The specific site was located in the bottom of a large sink approximately 2 acres in size which sloped gently to the east. The goldenseal population covered approximately one-fourth of the bottom area and extended 25 feet up the southern wall. Surface rock outcroppings were Mississippian aged limestones and conglomerates of the Monteagle formation. Tree canopy in the area was primarily tulip poplar, hickory, and maple with an occasional white or red oak.

The Walker county site was the largest visited, consisting of an estimated 10,000 plants. Approximately 25% of the plants were producing fruit. These plants appeared to be vigorous and no damage from pests or disease were detected.

Site # 2 was located in a private nature preserve in Hall county, Georgia just east of the Brevard Fault at the head of the Oconee river basin. We were accompanied to the site by the Supervisor and Head Biologist of the preserve. The goldenseal populations at this site were located in the bottom and 10 to 15 feet up the slopes of a steep ravine, draining to the southwest. Surface rock outcroppings were Pre-Cambrian Gneiss and Schists. These populations were in small groups of 10 to 100 plants each. Approximately 10% of the plants were producing fruit. They appeared to be vigorous and free of disease or pest damage. Tree canopy here was poplar, beech, and white oak.

Site # 3 was located in Overton county, Tenn. on private property, in the eastern section of the Western Highland Rim province of Tenn., just west of the Cumberland Plateau in the Cumberland River drainage basin. We were accompanied to the site by the landowner. The plants at this site were also in small batches of 10 to 100 plants each. These were the most vigorous of any of the other sites and approximately 50% of the plants were producing fruit. Canopy cover in the area was primarily poplar, beech, hickory, oak, and an occasional cedar.

Application of dolomite to the soil at the proposed site, at a rate of 18 pounds per 100 square feet, resulted in a significant increase in soil pH from 4.7 to 6.2 (Table 2). Consequently, the level of P, K, Ca, Mg, Cu, Fe, and Na increased from those observed in the sample taken in 2001. The Ca and Mg levels showed a marked increase from soil with pH 4.7 to a soil with a pH of 6.2. This enabled us to bring the soil pH and other parameters at SHF within the range of those soils from the native locations.

The areas to be used for the wild simulated certified organic goldenseal beds are first cleared of only as much undergrowth as necessary to allow the use of a roto-tiller to prepare beds and to effectively maintain the beds once established. A small tractor with backhoe attachment can be used for the removal of small stumps. Light values are measured in the open and under the canopy with a Sunfleck Ceptometer or like instrument. The values are compared and the larger hardwood trees pruned to achieve an average 66% shade.

TABLE 2 Soil Chemical Properties at Three Native Goldenseal Locations and Sleepy Hollow Farm. Location Sleepy Hollow Farm Amended May 31, May 31, Parameter Walker¹ Hall² Overton³ 2001 2002 pH 6.0 6.5 6.4 4.7 6.2 Organic Matter (%) 2.6 1.8 2.4 3.1 2.3 Phosphorus (ppm) 11 3 28 9 20 Potassium (ppm) 113 180 135 53 73 Calcium (ppm) 1549 1184 978 189 983 Magnesium (ppm) 101 204 72 47 334 Sulphur (ppm) 35 25 26 15 15 Boron (ppm) 0.6 0.8 0.8 0.3 0.4 Copper (ppm) 1.3 2.1 1.4 0.6 1.6 Iron (ppm) 75 69 129 118 124 Manganese (ppm) 319 138 181 124 128 Zinc (ppm) 4 3 20 2 2 Sodium (ppm) 20 19 19 17 23 CEC 11.8 10.9 7.2 7.1 10.4 CS - K % 2.5 4.2 5.3 2.1 1.8 Ca % 65.8 54.4 66.1 14.0 47.4 Mc % 7.2 15.6 8.3 5.8 26.8 H % 23.6 24.9 19.2 76.8 22.9 % Na 0.7 0.8 1.2 1.4 1.0 K:Mg Ratio 0.35 0.27 0.64 0.35 0.07 Sampling date: ¹May 17, 2001; ²Jun. 14, 2001; ³Jun. 19, 2001. 4: Mean comparisons between locations for sampling dates in 2001 only. CEC: Cation exchange capacity. CS: Cation saturation.

Once the area is prepared, beds 4 foot wide are roto-tilled to a depth of 4 to 6 inches and the soil amended with composted leaves and other organic additives, as described above, based on native soil test results. Native conditions are further simulated by raking the dry leaves and other debris back on the top of the soil after planting. Length of the beds is dependent upon topography and tree location.

Wild simulated certified organic goldenseal plantations can be established using either seed or, the preferred method, vegetative propagation such as rhizome division or in vitro tissue culture. An example of vegetative propagation by rhizome division is to divide one pound of goldenseal rhizomes by breaking or cutting into approximately 100-4.5 gram pieces. Each piece should have at least one growth bud and one attached root. These pieces are then planted in the beds described above at a depth of approximately 3 to 4 inches using 6 inch spacing in each direction yielding 4 plants per square foot. These plants are then allow to grow for a minimum of four years. Cultivation for a period of at least 4 years meets CITES regulations and permits export of the rhizomes outside the U.S.

Selective harvesting of the various plant parts based on whether the individual plant is reproductive or sterile permits standardization of the raw material by combining the parts in calculated amounts to arrive at pre-determined levels of alkaloid concentration. Table 3 lists the berberine/hydrastine levels of the various parts of goldenseal, rhizome, root, leaf, and stem cultivated using the preferred method. Table 4 lists the berberine/hydrastine levels cultivated outside of the parameters defined by the preferred method of this invention. In addition the alkaloid content of the various plant parts of seed producing plants is also compared with that of non-seed producing or sterile plants.

As reflected in the proposed USP standards for goldenseal powder, the berberine content of goldenseal is normally found to be greater than the content of hydrastine. However, as indicated by the tables below, our research has shown significant differences between not only the total alkaloid content of reproductive plants vs sterile plants but also in the ratio of berberine to hydrastine. The ratio of berberine to hydrastine is reversed in sterile plants. Sterile plants produce a greater amount of hydrastine compared to reproductive plants. In addition, the leaf produces a greater amount of hydrastine than berberine with sterile plants producing roughly 50% more than reproductive plants. Furthermore, hydrastine is generally below detectable levels in the stem.

TABLE 3 Alkaloid content of reproductive goldenseal plant parts compared with sterile plants soil pH > 6.0 Berberine % Hydrastine % Combined Dry weight Dry Weight Alkaloid % Reproductive Plant Part Rhizome 5.51% 4.85% 10.36% Root 3.58% 2.02% 5.60% Leaf 0.65% 0.93% 1.58% Stem 0.98% absent 0.98% Sterile Plant Part Rhizome 2.76% 3.17% 5.93% Root 0.99% 1.62% 2.61% Leaf 0.84% 1.44% 2.28% Stem 0.79% absent 0.79%

TABLE 4 Alkaloid content of reproductive goldenseal plant parts compared with sterile plants soil pH < 5.0 Berberine % Hydrastine % Combined Dry weight Dry Weight Alkaloid % Reproductive Plant Part Rhizome 3.24% 3.24% 6.48% Root 2.73% 1.96% 4.69% Leaf 1.28% 1.95% 3.23% Stem 1.20% absent 1.20% Sterile Plant Part Rhizome 1.98% 2.24% 4.22% Root 1.55% 0.80% 2.35% Leaf 0.68% 1.50% 2.18% Stem 0.99% 0.22% 1.21%

Current production practices make no differentiation between reproductive or sterile plants when harvesting however, based on the above data, it is apparent that by selective harvesting of goldenseal based on whether the individual plant is reproductive or sterile and separation of the various plant parts, the resulting raw material can be standardized to either a desired ratio of berberine to hydrastine or specific levels of both by a calculated mixture of the various parts and/or reproductive or sterile plants. This method will produce pharmaceutical grade goldenseal raw material (dry powder after grinding) suitable for use in teas, capsules, and/or tablet manufacture. The standardized powder is also forms an excellent starting point for standardized tincture or extract production.

It is very easy to differentiate between reproductive and sterile plants when harvesting since reproductive plants produce a forked stem with two leaves, a flower, and a bright red raspberry like fruit. Sterile plants do not produce forked stems and only produce one leaf per stem. Therefore selective harvesting of reproductive and sterile plants is the preferred method of harvesting goldenseal to be utilized in this invention.

Selective harvesting of the stem and leaf is relatively simple, first the plant is identified as being either reproductive or sterile as described above then the stem is cut (either manually or mechanically) at ground level. The leaf is separated from the stem by cutting or pulling, then the parts are placed and held in separate labeled containers until needed for determination of alkaloid content by one of the methods described below and then further processing. If the root and rhizome are not to be harvested at the same time, a colored marker, example red for reproductive plants and green for sterile, is placed in the bed location of the plant so it can be identified at a later time.

The rhizome and root are harvested either manually or mechanically by loosening the dirt surrounding the rhizome and root then removing them from the ground. The rhizome and root are identified as either reproductive or sterile then placed in labeled containers. After harvesting the goldenseal is prepared for drying by placing the material on wire mesh screens and washing with potable water to remove soil and debris.

Separation of the root and rhizome is accomplished by drying the material then placing the material in a cylindrical container. The container is rotated which tumbles the material similar to a clothes dryer. This tumbling causes the roots to break off the rhizome. The material is then screened using a mesh size which permits the smaller pieces of the root to pass through but retains the larger rhizome.

FIG. 2. illustrates the recovery rates from various methods of drying. There are significant differences in recovery between the sun dried and shade dried when compared to the material from the controlled drier, 32% and 33% vs. 27% respectively. This difference is attributed to more complete drying from the controlled drier. Scores from the visual appearance, texture, and odor ratings revealed material from the controlled drier to be the most desirable, followed by the sun dried with the shade dried being the least desirable. The control dried material has a darker more distinct color, a firmer snap, and a slightly stronger odor than the other two methods. The sun dried material is very close to the controlled dried with the exception that the color is somewhat lighter. The shade dried material is less firm and noticeably lighter in color.

TABLE 5 Total Hydrastine % Berberine % Total % Grams 1 lb. Fresh 1.05% 1.04% 2.09% 9.48 Sun Dried 3.34% 3.82% 7.16% 10.39 Shade Dried 3.17% 3.44% 6.61% 9.90 Controlled Drier 3.24% 3.78% 7.02% 8.60

The sun dried and the controlled drier produces total hydrastine/berberine percentages which are not significantly different however, there are significant differences between both the sundried (7.16%) and the control dried (7.02%) goldenseal and the material which was shade dried (6.61%). This percentage was then applied to the recovery factor from the drying methods in order to determine the total grams of alkaloids produced from each pound of fresh goldenseal. Even though the control dried material compared favorably with the sun dried material in terms of percentage of combined alkaloids, the actual amount of alkaloids produced from one pound of fresh goldenseal was significantly greater in the sun dried samples 10.39 grams vs. 8.60 grams from the control dried samples, a 17% difference. The sun dried material produced the greatest amount of hydrastine and berberine therefore sun dried material is the preferred method of drying for this invention.

TABLE 6 Sun Shade Controlled Dried Dried Drier Ground Dry 7.50% 6.78% 3.37% Ground Frozen 4.91% 4.12% 2.71% Ground Frozen/Thawed 4.38% 3.77% 3.17%

Table 6 details the percentage of raw material lost during the grinding process. There were significant differences between the various methods of grinding with the material from the controlled drier posting the least amount of loss in each method of grinding with the control dried ground frozen category performing best with a loss of 2.71% and the sun dried ground dry category the worst with a 7.50% loss. The rather high results posted by the sun and shade dried, ground dry material can most likely be attributed to mill operator inexperience however, even when allowing for this the controlled drier material still significantly outperformed the other drying methods, exhibiting less tendency for caking and packing while producing a somewhat brighter color end product.

TABLE 7 Sun Shade Controlled Dried Dried Drier Dry 7.12% 6.82% 6.96% Frozen 7.21% 6.95% 7.65% Froz/Thaw 7.16% 6.64% 7.46%

Table 7 contains a listing of the total hydrastine/berberine concentration in each sample after the grinding process was complete. Grinding while frozen consistently produced a higher percentage of alkaloid content than the other treatments. The frozen and frozen/thawed treatments produced a greater total alkaloid percentage in the sun dried and control dried categories than the dry material which was ground without any additional treatment. The control dry frozen sample produced the highest percentage alkaloid content, 7.65%, while the shade dried frozen/thawed sample was the lowest at 6.64%.

TABLE 8 Sun Shade Controlled Dried Dried Drier Fresh Before Grinding 10.39 gr 9.90 gr 8.60 gr 9.48 gr Grnd. Dry 9.55 gr 9.52 gr 8.23 gr Grnd. Frozen 9.95 gr 9.97 gr 9.11 gr Grnd Froz/Thaw 9.93 gr 9.56 gr 8.84 gr

Table # 8 displays the net effect of the various treatments on the amount of hydrastine/berberine available for extraction from one pound of fresh goldenseal. This was calculated by multiplying the weight of the fresh material times the percentage of recovery after drying times the percentage of recovery after grinding times the percentage of alkaloid content of the final product. The sun dried and shade dried methods produced results which were not significantly different however, both were significantly better than the results produced by the controlled drier method. Shade dried ground frozen produced the greatest amount of alkaloids per pound of starting material but it was not significantly different from the other shade or sun dried treatments. The controlled drier performed the poorest. FIG. 4 is a visual presentation of those results when compared with starting fresh material. Therefore grinding while frozen is the preferred method of grinding goldenseal for this invention.

Purchased powdered goldenseal, HPLC 2.624% hydrastine, 3.269% berberine, was used to develop the liquid extraction method. FIG. 4 visually illustrates the relative amounts of combined hydrastine/berberine recovered from the above goldenseal powder using solvents with five different concentrations of 95% ethanol and water. Total alkaloid recovery was greatest from the 1:3:1 (goldenseal:95% ethanol:water) solvent mixture and lowest from the 1:0:4 or 100% water solvent. An optimum concentration level was apparent at approximately 60% ethanol.

TABLE 10 Optimal Solvent Concentration Ethanol:Water % Alk % Alk % Alk Concentration Ext 1 Ext 2 Ext 3 4:0 0.775 0.302 0.322 3:1 1.144 0.397 0.244 2:2 1.102 0.361 0.171 1:3 0.844 0.354 0.135 0:4 0.615 0.322 0.213

The second extraction produced a somewhat flatter curve than the first. The 1:3:1 solvent mixture again produced the highest total concentration of combined alkaloids. The third extraction, in which the solvent concentrations were reversed, was designed to capture any alkaloids not recovered by its reciprocal concentration. The 1:4:0 solvent concentration is preferred for the third extraction. The overall results indicated that the solvent concentration levels with the lesser amounts of ethanol recovered a lower percentage of alkaloids except for the 1:4:0 concentration level which was not a great as the 1:3:1 level with an approximately 60% ethanol concentration appearing to be the optimal.

This concentration level was then used to produce a liquid goldenseal extract from goldenseal which had been sun dried and ground while frozen. The beginning alkaloid content of this material as measured by HPLC was 3.337% hydrastine and 3.822% berberine for a combined total of 7.159%. Utilizing the same procedure as above 12 ounces of liquid goldenseal extract was produced and spectrophotometrically determined on-farm to contain 0.62% hydrastine and 0.60% berberine, total 1.22%. This crude extract was then concentrated 3:1 and reanalyzed revealing a 1.89% hydrastine and 1.72% berberine content, total 3.61%.

Data from the above experiments was analyzed and the following table constructed to illustrate the relative cost effectiveness of each solvent concentration. The

TABLE 11 Extraction Cost Optimazation Ethanol: ozs.@ Water Total % Total % Total % Combined 1% Cost for Cost for Total Cost Conc. Ext 1 Ext 2 Ext 3 % 12 ozs. Alkaloids etoh/h₂o goldenseal Cost oz./1% 4:0 0.775% 0.302% 0.322% 0.466% 5.59 $0.53 $4.00 $4.53 $0.81 3:1 1.144% 0.397% 0.244% 0.595% 7.14 $0.67 $4.00 $4.67 $0.65 2:2 1.102% 0.361% 0.171% 0.545% 6.54 $0.61 $4.00 $4.61 $0.71 1:3 0.844% 0.354% 0.135% 0.444% 5.33 $0.50 $4.00 $4.50 $0.84 0:4 0.615% 0.322% 0.213% 0.383% 4.60 $0.43 $4.00 $4.43 $0.96 solvent from each of the 5 concentration levels used for the initial extraction was combined with the respective solvent from the other two extractions yielding 12 ounces of liquid extract containing a combined hydrastine/berberine concentration as shown in the table above. This number was then converted to the number of ounces of extract if it contained 1% alkaloids. Cost for the ethanol/water used was calculated based on actual cost, maintaining a 25% ethanol level after concentration as a preservative. The cost for goldenseal was the price we paid for the goldenseal used to develop the process, $64 per pound. The combination of these two factors produced a total cost for the batch. That number was then divided by the number of ounces @ 1% alkaloids for each batch to produce the final cost per ounce for each of the five ethanol/water concentration levels.

TABLE 12 Final Cost Cost per Ethanol:Water Combined ozs.@ Cost for Cost for Total oz. per Concentration % 12 ozs. 1% Alkaloids etoh/h₂o goldenseal Cost 1% 3:2 0.600 7.20 $0.68 $4.47 $5.15 $0.72

This mode was then used to develop a cost per ounce of raw materials used to produce the liquid extract from Sleepy Hollow Farm's material. The cost for the goldenseal was based on a price of $10 per 1% alkaloid content per pound. We believe this method of pricing to be much more representative of the value of any particular goldenseal lot for both the grower and the bulk buyer. This is equivalent to the current market for cultivated goldenseal of $35 per pound with a 3.5% minimum alkaloid content and yields a cost per pound slightly more than that paid for the previous material, $71.59/lb for SHF goldenseal compared with $64/lb from a distributor. The final cost per ounce of liquid goldenseal extract containing 1% alkaloids produced from Sleepy Hollow Farm's harvest was comparable with that from the purchased material even though the assumed cost per pound was higher due to its higher alkaloid content.

To summarize the method, powdered goldenseal, produced as described above utilizing wild simulated cultivation methods certified as meeting the standards of the NOP by a third party certifier authorized by the USDA, is sun dried then ground while frozen. The powdered goldenseal is then combined with a solvent mixture consisting of about 60% certified organic grain alcohol (95% ethanol) and about 40% water in a ratio of 1 part goldenseal powder by weight to 4 parts solvent mixture by volume in a suitable container. The container is constantly agitated by either shaking or using a motorized mixer with paddles attached which rotate at a speed of approximately 60 to 120 rounds per minute for 24 to 72 hours. The mixture is then filtered and the liquid reserved in a separate labeled amber glass container while the once extracted goldenseal is returned to the extraction container. The container is then refilled with the same quantity of the ethanol/water mixture as before and the procedure repeated. The mixture is filtered a second time and the liquid again reserved in a separate labeled container. The twice extracted goldenseal is then returned to the extraction container which is refilled with fresh solvent mixture containing 100% certified organic grain alcohol (95% ethanol). The process is repeated, then mixture filtered for a third time and the liquid reserved in a separate labeled container.

Current state of the art would, at this point, combine the solvent mixtures from the above extractions, take samples of the combined mixture and subject them to HPLC analysis, then dilute or concentrate the mixture in order to obtain a desired level of either berberine, hydrastine, or a combination of both expressed as “total alkaloids”. The method of this invention is through an added step in the process which will allow standardization to specific levels of both berberine and hydrastine. Details of that additional step follow:

The crude extracts from the above process are combined, samples taken and HPLC analysis performed, and then concentrated 3:1 using a water bath evaporator at approximately 40 EC and vacuum of 20 inches Hg. This concentration removes all the ethanol from the mixture. Since hydrastine is not very soluble in water it begins to precipitate out of the mixture once the ethanol is removed while the berberine remains in solution. This precipitation is monitored and once the hydrastine level reaches a predetermined point, non-limiting example, exactly 2 parts berberine/1 part hydrastine, the mixture is decanted and then diluted with certified organic grain alcohol (95% ethanol) to obtain the desired level of standardization. If a hydrastine rich product is desired, 1 part berberine/2 parts hydrastine as a non-limiting example, the hydrastine is allowed to precipitate to a predetermined point, then a calculated percentage of the berberine rich solution is decanted, the remaining mixture containing the hydrastine precipitate is then diluted with a calculated volume of certified organic grain alcohol (95% ethanol), the mixture thoroughly blended and the hydrastine precipitate reintroduced into the mixture. If a solid extract is desired, the mixture would then be evaporated to dryness using a water bath evaporator, spray drying, or other method of drying. The resulting product can be very precisely standardized to the content of both hydrastine and berberine.

The method permits the preparation of a pharmaceutical grade combination of hydrastine and berberine, where the practitioner or researcher selects the relative proportions of the two ingredients. This process is a major step up in the quality of goldenseal tinctures and extracts and can be used to produce pharmaceutically accurate combinations of active ingredients from goldenseal and other plants which have medicinal value.

A schematic diagram for goldenseal standardized extract process is presented in the drawing section. This is intended only for illustration purposes of the several aspects of the invention.

HPLC Method of Hydrastine/Berberine Determination in Goldenseal.

Principle: Hydrastine and Berberine, two major alkaloids in goldenseal are extracted from herbs and herb extracts using acidic aqueous methanol solution. They are then quantitated by isocratic HPLC at 235 nm using a reversed phase column.

Equipment: HP 1100 Chromatograph (or equivalent) equipped with the following:

Autosampler.

UV Detector capable of 235 nm.

Column-Luna phenyl-hexyl 150*4.6 mm, 5_M, Phenomenex

Data Acquisition-HP Software or equivalent.

Materials:

Acetonitrile—HPLC grade (EM Science).

Isopropanol—HPLC grade (EM Science).

Phosphoric acid 85% (Fisher)

Water HPLC grade (Fisher Scientific).

Extraction solvent: 50% methanol acidic solution: mix 250 ml methanol and 250 ml water, and 10 mL concentrated HCl.

Sodium Dodecyl Sulfate, HPLC grade from Fisher.

(1R,9S)-β-hydrastine standard from Sigma Chemical Co.

Berberine standard from Sigma Chemical Co.

Mobile Phase: 40% acetonitrile+10% Isopropanol+0.2% phosphoric acid+5mM Sodium Dodecyl Sulfate.

To prepare 1 Liter, measure ˜400 mL HPLC grade water in a 1000 mL graduated cylinder and add 1.44 g Dodecyl Sodium Sulfate to it. In a separate 500 mL graduated cylinder, measure 400 mL of acetonitrile and add to the solution. In a separate 100 mL graduated cylinder, measure 100 mL of isopropanol and add to this solution. Add 2 mL phosphoric acid to this solution. Dilute to volume with HPLC grade water and mix well. HPLC Condition: The following HPLC conditions were used when carrying out this analysis: Instrumental Assay Parameters: Flow Rate: 1.2 mL/min. Wavelength: 235 nm. Injection Volume: 10 μL. Run Time: 12 min. Hydrastine Elution Time—Approximately 4.0 minutes. Berberine Elution time B Approximately 7.6 minutes Preparation of Standard Solution:

Accurately weigh about 15 mg Hydrastine chloride and 15 mg Berberine chloride into 25 mL volumetric flask, record the weight (important: correcting for water and HCl) Add approximately 15 mL 50% methanol acidic solution and sonicate for 15 minutes. Allow the flask to cool to room temperature and fill to full volume with 50% methanol acidic solution.

Measure 5 mL above solution and transfer to a 25 mL volumetric flask and diluted to the full volume using 50% methanol acidic solution (standard solution).

Preparation of Samples:

For herbs, accurately weigh about 40 mg powder into a 15 mL centrifuge tube

Add 14 mL of 50% methanol acidic solution and sonicate for 30 min.

Centrifuge for 10 minutes. Put 1 mL clear solution into HPLC vial for analysis

For liquid sample, Pipet 0.5 mL into 15 mL volumetric flask, dilute to 14 mL using extraction solution.

Centrifuge for 10 minutes. Put 1 mL clear solution into HPLC vial for analysis

Calculation and Reporting of Results:

The two alkaloid contents in a sample are calculated using the following equation are reported as each individual one:

alkaloid=

Pu=Peak area of each alkaloid in the sample.

Ps=Peak area of alkaloid in the standard.

Cs=concentration of each free alkaloid in the standard (g/mL).

Wu=Weight of the sample (g).

50=volume of sample (mL), 100=converts to percent.

Alternative UV/Vis Spectrophotometric Method

Two buffer solutions were prepared by mixing appropriate amounts of citric acid (Fisher) and dibasic sodium phosphate (Fisher) in de-ionized ultra-filtered water (DIUF) (Fisher) to achieve pH of 5.6 and 7.2.

A berberine stock solution was prepared by dissolving 0.25 g of berberine hydrochloride hydrate 97% (Fisher) in 100 ml DIUF water. A berberine reference solution (1.25 mg %) was prepared by diluting 0.5 ml of the stock solution to 100 ml with 1 N HCl (Fisher). A hydrastine stock solution was prepared by dissolving 0.2 g (1R,9S)-β-hydrastine (Sigma) in 100 ml 1 N HCl. A hydrastine reference solution (4 mg %) was prepared by diluting 2 ml of the stock solution to 100 ml with 1 N HCl. A bromocresol purple solution was prepared by dissolving 50 mg of the dye (Fisher) in a few drops of 0.1 N HCL and bringing the solution to 100 ml with DIUF water. Berberine Determination: 0.5 ml of the crude goldenseal extract, prepared using the process developed above, is diluted to 100 ml with pH 7.2 buffer. A 10 ml aliquot is transferred to a 60 ml separator containing 10 ml chloroform (Fisher) and 1 ml of the bromocresol purple dye solution. The mixture is shaken for 1 minute and the layers allowed to separate completely. The chloroform layer is transferred to a second 60 ml separator and the remaining aqueous layer re-extracted with an additional 10 ml chloroform. The combined chloroform extracts are shaken for 15 seconds with 10 ml 0.1 N NaOH (Fisher) to release the combined dye. The aqueous layer is then transferred to a beaker and diluted to 25 ml with DIUF water. A sample of the final solution is transferred to a 1 cm cell and placed into a Genesys 10 UV/Vis spectrophotometer with additional cells containing the berberine reference solution and a blank containing 1 N HCl. The absorbencies are measured at 590 nm and recorded. The concentrated (3:1) goldenseal extract is analyzed for berberine in the same manner as above except 3 ml of the dye solution is used and the final solution diluted to 50 ml with DIUF water in order to get a reading within the limits of the machine. Hydrastine determination: 1.0 ml of the crude goldenseal extract, prepared using the process developed above, is diluted to 100 ml with pH 5.6 buffer. A 10 ml aliquot is transferred to a 60 ml separator containing 10 ml chloroform (Fisher) and 1 ml of the bromocresol purple dye solution. The mixture then shaken for 1 minute and the layers allowed to separate completely. The chloroform layer is transferred to a second 60 ml separator and the extraction repeated with 2×10 ml chloroform. The combined chloroform extracts are shaken for 15 seconds with 10 ml 0.1 N NaOH (Fisher) followed by 5 ml saturated sodium chloride solution (Fisher). The chloroform layer is then evaporated just to dryness. The residue is dissolved in 1 N HCl, transferred to beaker and diluted to 25 ml. The absorbencies of the final solution together with the hydrastine reference solution is measured concomitantly in 1-cm cells at 345 nm with Genesys 10 UV/Vis spectrophotometer using 1 N HCl as a blank. The concentrated (3:1) goldenseal extract is analyzed for hydrastine in the same manner as above except 3 ml of the dye solution is used and the final solution is diluted to 50 ml with DIUF water in order to get a reading within the limits of the machine. Standardization to Biological Activity

The following is an example of a method for standardizing the mixture of pharmacologically active mixtures of chemical components to antimicrobial activity. The example is intended only for illustration purposes of the several aspects of the invention.

Briefly, a sample from a combined extract lot as prepared above is taken and subjected to HPLC analysis to determine the berberine concentration therein. Another sample is taken and used to determine the minimum inhibitory concentration (MIC) and/or minimum bactericidal concentration (MBC) of the crude extract against a specific microorganism. MIC and/or MBC for a berberine reference standard is obtained at the same time. The procedures for making this determination are detailed after the following discussion. The results of these analyses are recorded.

The MIC (or MBC) of the berberine reference standard is divided by the MIC (or MBC) of the crude extract resulting in an “Activity Factor”. An “Activity Factor” of 1 means the activity of the crude extract and the reference standard are equal, greater than 1 means the crude extract has greater activity than the standard and less than 1 means the crude extract has a lesser activity than the standard. (See Formula 1 below) The “Activity Factor” can be used as a standard to compare the activity of different batches or other manufacturers products against the same organism.

The reciprocal of formula 1, MIC (or MBC) of the crude extract divided by the MIC (or MBC) of the berberine reference standard yields a “Catalyst Factor”. This number quantifies the effect of the unknown compounds which enhance the activity of berberine and is used in conjunction with the HPLC analysis of the crude extract to calculate the concentration of berberine in the crude extract necessary to produce activity equivalent to the reference standard or a fraction or multiple thereof as desired. (See Formula 2 below)

Once the concentration of berberine in the crude extract necessary to equal the activity of the standard is known, the crude extract can be standardized to a desired “Activity Factor” by manipulating the berberine level to a desired multiple or fraction of the standard.

CF=Catalyst Factor, quantifier of the effects of the unknown compounds

AF=Activity Factor, berberine reference standard=1

AF_(target)=Desired Activity Factor

MIC_(extract)=MIC of the crude extract against a specific organism, μg/ml

MIC_(berberine)=MIC of the berberine reference standard against a specific organism,

μg/ml

%_(berberine)=Concentration of berberine in the crude extract

%_(required)=Concentation of berberine required to equal the activity of berberine reference standard

%_(final)=Concentration of berberine in final product required to produce AF_(target) (MIC_(berberine)/MIC_(extract))=AF  Formula 1 Example data from Hwang (2003) MIC of berberine and crude goldenseal root and rhizome extract against Streptococcus mutans 125/250=0.5 (MIC_(extract)/MIC_(berberine))=CF  Formula 2 250/125=2 CF×%_(berberine)=%_(required)  Formula 3 2×0.02%=0.04% berberine concentration in the crude extract required to equal the biological activity of the berberine reference standard. AF_(target)×%_(required)=%_(final)  Formula 4 Assuming an arbitrary AF_(target) of 5 times the standard: 5×0.04%=0.20% final berberine concentration to yield a product with 5 times the activity of the reference standard.

The extract is then standardized to the desired Activity Factor by manipulating the level of berberine using the process as previously described in the above examples.

The same procedure can be used to standardize to other pharmacological activities by substituting the appropriate test of pharmacological activity for the MIC/MBC tests.

Production follows the same procedure as previously described up to the point where the solvent mixture from each of the three extractions are blended into a single batch and before standardization of alkaloid content step.

Antimicrobial Activity Determination

a. Minimum Inhibitory Concentration (MIC) of Goldenseal Extract Preparations Against Selected Human and Animal Pathogens

Minimum inhibition concentration (MIC) will be tested in appropriate liquid media in 96-well microtiter plates. Each well contains 5×10⁵ CFU/ml of test bacteria, serially diluted extracts and medium. In some cases when chemical solvents are used, it will be tested simultaneously with the test compounds to ensure the absence of antimicrobial activity. Triplicate samples are to be performed. All plates will be incubated at 37° C. and growth estimated spectrophotometrically (660 nm) after 48 hr using a microplate reader (molecular device, Vmax kinetics, Menlo Park, Calif.). The MIC for each test bacteria is defined as the minimum concentration of test compound limiting turbidity to <0.05 absorbance at 660 nm. b. Minimum Bactericidal Concentration of Goldenseal Extract Preparations Test bacteria will be exposed to goldenseal extract at MIC, 2×MIC, and 5×MIC concentration. After 10 min at 37° C., the treated bacterial (or fungal) culture will be diluted and viable colony counts determined. The ability of goldenseal extract in killing test organisms will be elucidated. The concentration capable of reducing 99.99% viable organism will be noted as MBC. Alternative Method of Manipulating the Concentration Level the Pharmacologically Active Compounds In Goldenseal Extracts

An alternative method of arriving at a desired concentration level of pharmacologically active compounds in goldenseal extracts begins with raw goldenseal cultivated using the preferred method, selectively harvested based or whether the plants are reproductive or sterile, then separating the various parts of each. The various plant parts from the reproductive plants and the various plant parts from the sterile plants are individually processed by drying, grinding, and extraction as previously described. Before the individual extracts are combined, samples are taken from each and quantitatively analyzed for berberine/hydrastine content using one of the analytical methods previously described. These extracts will contain various ratios of berberine/hydrastine depending upon which part is extracted and the stage of extraction, first, second, or third. These extracts are then combined in calculated amounts to produce the desired levels and ratios of berberine/hydrastine or further concentrated and standardized using the preferred method.

Second Alternative Method of Manipulating the Concentration Level the Pharmacologically Active Compounds in Goldenseal Extracts

A second alternative method of arriving at a desired concentration level of pharmacologically active compounds in goldenseal extracts begins with raw goldenseal cultivated using the preferred method, selectively harvested based or whether the plants are reproductive or sterile, then separating the various parts of each. The various plant parts from the reproductive plants and the various plant parts from the sterile plants are individually processed by drying and grinding as previously described. The resulting powders are individually quantitatively analyzed for berberine/hydrastine content using one of the analytical methods previously described. The powders are then blended in calculated amounts to produce a composition to be used for extraction which has desired ratios and levels of berberine/hydrastine.

This pre-standardized powder is then extracted using the previously described method. The compositions from the 3 extraction stages are then either combined, analyzed and standardized to a desired level and/or ratio of berberine/hydrastine as described in the preferred method or individually analyzed, then combined to produce a desired level and/or ratio of berberine/hydrastine as described in the first alternative method.

The methods of the present invention are expected to have most widespread application in differentiating, improving and achieving reproducibility or standardization of herbal processing techniques, particularly extraction of pharmacologically active mixtures from plant sources and in obtaining plant extracts of reliable pharmacological activity. The reproducibility and standardization procedures of the invention involve the use of a combination of pharmacological and chemical analysis of the isolated products, typically obtained in extraction procedures.

The pharmacological tests performed on the process products, preferably plant extracts, most preferably the plant extract obtained from goldenseal, may be in the form of in vitro and/or in vivo pharmacological tests. In the present invention it is preferred that at least two in vitro and at least two in vivo pharmacological tests be used. These tests are generally correlated with a changed biological state of a living organism. This may take the form of either an enhanced condition of the organism or an effective treatment of a medical condition in a patient.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Reference is made to standard textbooks and other references (e.g., journal articles) that contain definitions and methods and means for carrying out basic techniques, encompassed by the present invention.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

REFERENCES

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1. A method of reproducibly obtaining a desired level of pharmacological activity from pharmacologically active extracts derived solely from a plant selected from the genus Hydrastis comprising: (a) conducting a plurality of different extraction processes on a plurality of samples from the same plant of the genus Hydrastis to obtain a plurality of crude plant extracts; (b) conducting at least one biological test on each of the crude plant extracts to determine the level of pharmacological activity thereof, wherein said test is known to correlate with a medical condition of a living organism; and (c) combining 2 or more of the extracts from step (b) to obtain a specific or desired level of pharmacological activity in the combination of extracts to provide a standardized product having the desired level of pharmacological activity.
 2. The method of claim 1 wherein the at least one test is at least one of an in vitro test and/or at least one of an in vivo test.
 3. The method of claim 2 wherein the at least one test is at least two in vitro and at least two in vivo tests.
 4. The method of claim 3 wherein the organism is a human or animal.
 5. A method of reproducibly obtaining a pharmacologically active plant extracts, having a desired level of pharmacological activity, derived solely from a plant selected from the genus Hydrastis, comprising: (a) conducting a plurality of different extraction processes on a plurality of samples from the same plant of the genus Hydrastis to obtain a plurality of crude plant extracts; (b) conducting at least two in vitro and at least two in vivo biological tests on each of the plurality of the crude extracts to determine the level of pharmacological activity of each extract thereof, wherein the test is known to correlate with effective treatment of a medical condition in a patient, and (c) combining two or more of the extract from step (b) to obtain the desired level of pharmacological activity in the combined extracts to provide a standardized product having the desired level of pharmacological activity.
 6. The method of claim 1, wherein the Hydrastis is Hydrastis canadensis L. (goldenseal).
 7. The method of claim 5, wherein the plant material is ground prior to extraction.
 8. The method of claim 7, wherein the plant material is ground to a mesh size between about 10 and 80 mesh.
 9. The method of claim 5, wherein the extraction step(s) is performed using a mixture of extraction solvents.
 10. The method of claim 5, wherein the extraction step(s) is performed using a single solvent or a mixture of two or more of the solvents selected from the group consisting of acetone, acetonitrile, dichloromethane, ethyl acetate, ethanol, hexane, isopropanol, methanol, other alcohols, water, supercritical carbon dioxide and mixtures thereof.
 11. The method of claim 5, wherein the Hydrastis is Hydrastis canadensis L. (goldenseal).
 12. A method of reproducibly obtaining a desired level of pharmacological activity from pharmacologically extracts derived solely from a plant selected from the specie Hydrastis canadensis L. (goldenseal), comprising: (a) conducting a plurality of different extraction processes on a plurality of samples from the same plant of the Hydrastis canadensis L. (goldenseal) to obtain a plurality of crude plant extracts; (b) conducting at least one biological test on each of the plant extracts to determine the level of pharmacological activity thereof, wherein said test is known to correlate with a medical condition of a living organism; and (c) combining two or more of the extracts from step (b) to obtain a specific or desired level of pharmacological activity in the combined extracts to provide a standardized product having the desired level of pharmacological activity. 